DESCRIPTION (adapted from the application) Iron is essential for normal metabolic functions and disturbances of iron homeostasis can have significant clinical consequences. The amount of iron in the body is controlled at the point of absorption in the small intestine, yet the mechanism by which iron traverses the intestinal epithelium is poorly understood. In this application we aim to study in detail the mechanism of iron movement out of the epithelial cells and into the body by investigating the biology of two recently cloned genes. One of these, hephaestin, is known to play a role in iron export from the epithelium, while the other, ireg1, is proposed to play an important role in this process. We hypothesize that hephaestin and ireg1 interact to form a complex which transports iron across the basolateral membrane of intestinal enterocytes and delivers it to apotransferrin. This hypothesis will be tested by addressing several key questions: (1) Do hephaestin and ireg1 co-localise and physically interact in epithelia] cells? (2) Is this interaction sufficient to mediate iron movement across cellular membranes? (3) Can this complex directly or indirectly deliver iron to apotransferrin? and (4) How is the iron transport mediated by these proteins regulated? These issues will be addressed by conducting parallel studies using both intestinal tissue and cell lines overexpressing hephaestin and ireg1 and by studying iron transport in animal models. Immunomicroscopy, co-precipitation and the yeast two-hybrid system will be used to search for interactions between the two proteins. The role of hephaestin and ireg1 in iron transport will be studied by developing iron efflux assays in mammalian and yeast cells, and by the targeted disruption of the ireg1 gene in mice and the analysis of its phenotype. To determine whether the hephaestin/ireg1 complex delivers iron to apotransferrin, we will search for direct interactions between the proteins and will investigate the ability of apotransferrin to facilitate iron release from intestinal tissue and in a reconstituted iron efflux system. The iron-dependent regulation of ireg1 and hephaestin will be studied in cellular and animal models and a detailed analysis of the regulatory system will be carried out. In all studies, site directed mutagenesis will be used to dissect interactions at the molecular level. The iron efflux or transfer step of intestinal iron absorption has proved difficult to study in the past yet it is likely to be the rate-limiting for absorption under certain circumstances. However, the identification of hephaestin and ireg1 provides an unprecedented opportunity to investigate this process at the molecular level and to evaluate its role in human disorders of iron metabolism such as hereditary hemochromatosis and provides potential therapeutic targets.